Molecular diagnosis of Papaya meleira virus (PMeV) from leaf samples of Carica papaya L. using conventional and real-time RT-PCR.

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ContentslistsavailableatSciVerseScienceDirect

Journal

of

Virological

Methods

j o ur na l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j v i r o m e t

Molecular

diagnosis

of

Papaya

meleira

virus

(PMeV)

from

leaf

samples

of

Carica

papaya

L. using

conventional

and

real-time

RT-PCR

Paolla

M.V.

Abreu

a

_,

_João

_G.

_Piccin

a

_,

_Silas

_P.

_Rodrigues

a

_,

_David

_S.

_Buss

a

_,

_José

_A.

_Ventura

a,b

_,

Patricia

M.B.

Fernandes

a,∗

a_Núcleo_de_{Biotecnologia,}_Universidade_Federal_do_Espírito_Santo,_Av._Marechal_Campos,_1468,_29040-090,_Vitória,_ES,_Brazil b_Instituto_Capixaba_de_Pesquisa,_Assistência_Técnica_e_Extensão_Rural,_Rua_Afonso_Sarlo_160,_29052-010,_Vitória,_ES,_Brazil

Articlehistory: Received15July2011

Receivedinrevisedform1December2011 Accepted6December2011

Available online 16 December 2011

Keywords: Papayameleiravirus Papaya Resistanceinduction Phytopathology dsRNA Real-timeRT-PCR

a

b

s

t

r

a

c

t

Papayameleiravirus(PMeV)isthecausalagentofpapayastickydisease.Thisstudydescribestwomethods formoleculardiagnosisofPMeVusingconventionalandreal-timePCR.Thesemethodswereshowntobe moreefficientthancurrentmethodsofviraldetectionusingextractionofPMeVdsRNAandobservation ofsymptomsinthefield.Themethodsdescribedherewereusedtoevaluatetheeffectofinoculationof papayaplantswithpurifiedPMeVdsRNAontheprogressofPMeVinfection.Asingleinoculationwith PMeVdsRNAwasobservedtodelaytheprogressofthevirusinfectionbyseveralweeks.Thepossibility ofverticaltransmissionofPMeVwasalsoinvestigated.NoevidencewasfoundforPMeVtransmission throughseedscollectedfromdiseasedfruit.Theimplicationsoftheseresultsfortheepidemiologyof PMeVandthemanagementofpapayastickydiseasearediscussed.

1. Introduction

Papayastickydisease,or meleira,isanimportantand grow-ingprobleminpapaya(Caricapapaya L.)cultivation,capableof causingcompletelossofthecrop.Thetissuesofpapayacontain lactifers which are maintainedunder high pressure and exude latexuponinjury.Thislatexhashighlevelsofproteasesand alka-loids(ElMoussaouietal.,2001)andisbelievedtobepartofthe defencemechanismsoftheplant.However,papayastickydiseased plantsarecharacterisedbyaspontaneouslyexudationofﬂuidand translucentlatexfromthefruitandleaves.Thelatexoxidisesafter atmosphericexposure,resultinginsmallnecroticlesionsonthe edgesofyoungleaves,andastickysubstanceonthefruit(Kitajima etal.,1993;Zambolimetal.,2003)thatmakesthemcommercially unacceptableforconsumption(Venturaetal.,2004).

Thecausalagentofpapayastickydiseasehasbeenidentiﬁed asPapaya meleiravirus(PMeV),a 12kbpdsRNAvirusfoundas 50nmsphericalparticlesininfectedtissue(Kitajimaetal.,1993; Zambolimetal.,2003).Unusuallyforaplantvirus,PMeVappears toresideprimarilyinlactifers,whereitmodiﬁespotassiumlevels

∗ Correspondingauthor.Tel.:+552733357348;fax:+552721227275. E-mail addresses: patricia.fernandes@pq.cnpq.br, pmbfernandes@gmail.com

(P.M.B.Fernandes).

andthustheosmoticbalance,leadingtoruptureofcellsandlatex exudation(Rodriguesetal.,2009c).

AsthereareasyetnoresistantcultivarstoPMeV,controlisby visualidentificationofdiseasedplantsandtheireradication (rogu-ing)(Venturaetal.,2004).Forunknownreasons,visualsymptoms ofmeleiraonlyappearafterflowering(Venturaetal.,2004).Even then,diagnosiscanbedifficultasthereisarelativelylong asymp-tomaticincubationperiodandtheearlysymptomscanbeconfused withotherconditions(Venturaetal.,2004).Thereisthusaneed foralternativediagnosticmethods.

TwomethodshavebeenpublishedfordetectionofPMeVby molecularmeans.Thefirstmethod(Rodriguesetal.,2005)applies nucleicacidspurifiedfromlatextoagarosegelsandvisualisesthe resultant12kbpviraldsRNAbandwithethidiumbromide. This hastheadvantageofrelativelylowcostandrapidturnover,but requireslargequantitiesoflatexandahightiterofvirusandsois unsuitableforearlystagesofinfectionorwhenlittleplant mate-rialisavailable.Thesecondmethod(Araujoetal.,2007)applies conventionalreversetranscriptionPCR(RT-PCR)tolatexdilutedin ammoniumcitrate.Thisrequireslesslatexandismoresensitive, butbecausethemethodlacksanucleicacidpurificationstep,latex proteinsremaininthePCRreaction.Papayalatexhasahigh con-centrationofproteins,especiallyproteases(Moutimetal.,1999), whichcaninterferewiththePCRreaction.Inaddition,astronglink betweenPMeVandlatexparticleshasbeenobserved(Rodrigues etal.,2009c).Furthermore,dsRNAvirusessuchasPMeVhavethe

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capacity,undercertainconditions,totranscribewithinthe cap-sidenvelope(Harrison,2007).Takentogetherthesefactorswould reducethechanceofvirusdetectionbyPCR.

Amoresensitiveandqualitativetechniqueforvirusdetection isreal-timeRT-PCR(VincelliandTisserat,2008).Measurementby realtimePCRisbasedontheincreaseinfluorescenceofdyes,e.g., SYBRGreen,whenconnectedtoadouble-strandedDNAsothat fluorescencerecordedateachPCRcyclereflectsthenumberofPCR productsgenerated duringamplification(Morrisonetal.,1998). Thisinturnreflectstheinitialquantityoftemplateinthesample. Real-timeRT-PCRhasbeenusedsuccessfullytodetectlowlevels ofvirusesinanumberofplants(Limetal.,2010;Parisietal.,2011). Aswellasincreasedsensitivity,thetechniqueallows simultane-ousanalysisoflargenumbersofsamplescomparedtogelbased systems,andautomatedanalysisoftheresults.

AfurtherprobleminPMeVcontrolisthelimitedinformation aboutthevirustransmission.ThecommonBrazilianwhiteﬂy Tri-aleurodesvariabilishasbeenruledoutasavector(Rodriguesetal., 2009b).It hasproveddifﬁculttoinfectpapaya maturetrees by mechanicalinjuryshortofdirectPMeVinjectionduetoexudation oflatexanditssimultaneouspolymerisationontheinjuredtissues (Rodriguesetal.,2009b).Bothcommercialandprivatepapaya pro-ductionarefromtreesgrownfromseeds.However,thevertical transmissionofPMeVhasnotyetbeenevaluated.

Apromisingdevelopmentinhorticulturalviruscontrolisthe conceptthatplantscaninitiatedefenceresponsesbasedonRNA silencing when challenged byviral RNA. The useof transgenic plantswhichconstituentlyexpressviralmaterialhasbeen success-fullydevelopedindifferentcrops(Scorzaetal.,2001;Tricolietal., 1995). Alternatively,mechanical inoculation of viraldsRNAhas inhibitedinfectionbythreevirusclassesintoNicotiana benthami-ana(Tenlladoetal.,2003)andsimilarresultshavebeenachieved withdsRNAasaspray(Ganetal.,2010).

Ourobjectivesinthisstudywereﬁrstlytodevelopimproved methodsforthedetectionofPMeVbyconventionalandreal-time RT-PCR.Thepossibilityofverticaltransmissionoftheviruswas investigatedusingconventionalRT-PCR.Theeffectsofthe inocula-tionofpuriﬁedPMeVdsRNAinpapayaplantswerealsoassessed asapreliminaryprospectforpapayastickydiseasecontrol.

2. Materialsandmethods

2.1. Plantmaterial

Samplesof papaya(cv.Golden)werecollectedatthe Exper-imentalFarm oftheInstituto Capixabade Pesquisa, Assistência TécnicaeExtensãoRural(INCAPER),Espírito SantoState,Brazil, withtheaidofanagriculturaltechnicianexperiencedinthevisual diagnosisofpapayastickydisease.Fortheexperimentsevaluating differentPMeVdiagnosismethods,theleavesandlatexsamples (n=32)weredividedinto4categories:(1)plants(n=10) symp-tomatic for sticky disease, (2) plants (n=8) with inconclusive diagnosisforstickydisease,(3)plants(n=10)asymptomaticbut closetosymptomaticplantsand(4)plants(n=4)asymptomatic locatedat12rowsfromsymptomaticplants.Latex(300␮L)was sampledfromeachplantusingasterilerazorbladeandmixed(1:1, v/v)with0.1McitratebufferpH5.0(Rodriguesetal.,2009a).Leaf samples(∼2g)werealsocollectedin15mLsterileFalcontubes.The sampleswerecooledoniceandthenucleicacidswereextracted onthesamedayandstoredat−20◦_C.

TheexperimentsevaluatingthetransmissionofPMeVthrough seedsusedseedscollectedfromdiseasedfruit.Papayaseedsfrom Piracicaba,SãoPauloState,wherethereisnoevidenceofpapaya stickydisease,were used asnegative controls.The seedswere washedwithwateranddetergenttoremovethesarcotesta,dried

at25◦Canddisinfectedusing70%ethanolfor10sand2%sodium hypochloritefor5min.Afterrinsing3×withdistilledwater,the seeds(5–8seeds/plate)weretransferred toa150mmPetridish containing180mLofsterile1%agar(Suppl.Fig.S1)andincubatedat 16hlight/30◦Cand8hdark/20◦C.15–20daysaftergermination, seedlingsfromoriginallydiseasedfruit(n=172)orthenegative control(n=187)werecollected. Theseedlings fromeachgroup werepooledandgroundtogetherinliquidnitrogenbeforenucleic acidextractionasdescribedbelow.

For the experiments involving co-inoculation of papaya seedlingswithPMeVandpuriﬁedPMeV-dsRNA,theplants(n=12) weregrowninagreenhousein8kgpotscontainingfertilisedsoil andwhitesand(3:1)for3–4monthsaftergermination.

2.2. Co-inoculationofpapayawithPMeVandpuriﬁedPMeV dsRNA

LatexfrompapayastickydiseasedplantswasusedasaPMeV inoculum source(Kitajima etal.,1993; Rodrigueset al.,2009b; Zambolimetal.,2003).Thepuriﬁed PMeVdsRNAwasobtained aspreviouslydescribed(Rodriguesetal.,2005).Theplants(n=3) weredividedinthreegroups:(1)Plantssimultaneouslyinoculated with20␮Lof(1:1,v/v)diseasedlatex/phosphatebufferpH7.0and 20␮LofpuriﬁedPMeVdsRNA(91.7ng␮L−1_),₍₂₎_plants_inoculated

with20␮LphosphatebufferpH7.0and(3)plantsinoculatedwith 20_␮LofPMeVinfectedlatex,diluted(1:1,v/v)inphosphatebuffer pH7.0.Thegroups2 and3wereusedasnegativeand positive controls,respectively.Theinoculationswereperformedintheleaf petiolesusingasterilesyringe.Aliquotsoflatexandleafwere col-lectedfromtheshootapexusingasterilebladeat7dayintervals during71days,anddiluted(1:1,v/v)insodiumcitratebufferpH 5.0(Rodriguesetal.,2009a).Allsampleswerestoredat−80◦_C_until

used.

2.3. Extractionandanalysisofpapayanucleicacids

MoleculardiagnosisofPMeV-dsRNAfrompapayalatexwas per-formedasdescribed(Rodriguesetal.,2005).Thenucleicacidswere extractedfrompapayaleaf(100mg)aspreviouslydescribed(Doyle andDoyle,1990),withmodiﬁcations.Afterextractionusingorganic solvents, thesupernatant was precipitatedwith600␮Lof cold ethanoland6␮Lof3MsodiumacetatepH5.2.Thesampleswere incubatedat−20◦_C_overnight,_centrifuged_12,000_×_g_for₅₀_min_at

4◦C.Thenucleicacidsinthepelletwerewashedwith70%ethanol, dried,andresuspendedinwaterforquantityandpurity assess-mentusingaNanodropND1000spectrophotometer.Thesamples weretreatedwithDNaseI(RQ1RNase-FreeDNase,Promega) fol-lowingthemanufacturer’sinstructions.ThepresenceofintactRNA wasconﬁrmedbyRT-PCRoftheactingene(Suppl.TableS1).The nucleicacidswereseparatedon1%TBEagarosegelsfor1.5hat 80V.Thegelsstainedwithethidiumbromide(15ngml−1)were recorded usingan imagecapturesystem L-HE-Pix/L-PixIMAGE (Cotia,Brazil).

2.4. DetectionofPMeVbyconventionalRT-PCR

The primers used in this study are listed in Suppl. Table S1. PMeVconv primers were designed using Primer3 (http://frodo.wi.mit.edu/primer3/) targeting a PMeV sequence homologoustoRNA-dependentRNApolymerases(RDRP)reported by(Araujoetal.,2007).Inthis report,theauthorshadusedthe softwareGene RunnerVersion 3.05(Hastings Software,Inc.)to designaprimer,CO5-3,targetingthePMeVsequence.CO5-3was alsotestedinourstudy.Actinprimersweredesignedaspreviously described(Santos,2005).

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Papaya RNA (500ng) was incubated at 96◦C for 3min to denaturethePMeVdsRNA(Zambolimetal.,2003).cDNAwas syn-thesisedusingcDNArandomhexamers(250ng␮L−1₎_and_M-MLV

ReverseTranscriptasekit(Promega)followingthemanufacturer’s instructions.AllPCRreactionsusedGoTaqFlexiDNAPolymerasekit (Promega)followingthespeciﬁcprogramsinaMastercycler Ther-mocycler(Eppendorf,Hauppauge,USA):PMeVconvprimers,94◦C for3min,32cyclesat94◦Cfor1min,61◦Cfor1minand72◦Cfor 1min,and72◦Cfor7min,Actinprimers,94◦Cfor4min,36cycles at94◦Cfor1min,60◦Cfor1.5minand72◦Cfor1minand72◦Cfor 5min,CO5-3primer,94◦Cfor3min,30cyclesat94◦Cfor1min, 57◦Cand72◦Cfor2minand72◦Cfor7min(Araujoetal.,2007).All reactionswereconductedinduplicateandthePCRfragmentswere separatedon1.0%or1.5%TBEagarosegels,asdescribedabove. 2.5. DiagnosisofPMeVbyreal-timeRT-PCR

Theprimersusedinthereal-timeRT-PCRreactions,PMeVreal (Suppl.TableS1),weredesignedusingPrimer3againtargetingthe PMeVRDRP(Araujoetal.,2007).Theprimerstargetingthe riboso-mal28Sgenewereasdescribed(Rojasetal.,2009).

ThepapayaRNAsamples(600ng)weremixedwith2_␮L ran-domhexamers50␮M,1␮L10mMdNTPmix(AppliedBiosystems, Carlsbad,USA)andDEPCwaterto12␮Lﬁnalvolume.Thesamples wereincubatedfor96◦Cfor3minandthecDNAsynthesisedusing aSuperScriptIIIkit(Invitrogen,Carlsbad,USA)followingthe man-ufacturer’sinstructions.Brieﬂy,eachsamplereceived4␮LSuper Script5×buffer,2␮LDTT(0.1M),0.1␮LRNaseOUT(40U␮L−1_),

1.3␮L DEPC water and 0.6␮L 5× Super Script III (200U␮L−1₎

totalling20␮L.Thesampleswereincubatedfor10minat25◦C, 50minat50◦Cand5minat85◦C.TheobtainedcDNA received 3.5␮LofcDNA,10␮LofSYBRGreenPCRKitMasterMix(Applied Biosystems,Carlsbad,USA)and6.5␮Lofasolutionof2.8mMof eachprimerdilutedin0.01MTrispH8.0.Themixturewas incu-bated at 95◦C for 10min,40 cycles at95◦C for 15sand 60◦C for1min,95◦C for15s, 60◦C for1minand95◦Cfor15susing a7500RealTimePCRSystemandthedataanalysedusingaSDS SoftwareSystem7500version2.0.1(AppliedBiosystems,Carlsbad, USA).Relativeabundancewasestimatedbychoosingathresholdof SYBRGreenfluorescenceduringtheexponentialphaseof fluores-cenceincrease.Thecyclenumberatwhicheachsamplepassesthe fluorescencethreshold(Ct)wasrecorded(Bustinetal.,2009).To preventfalsepositives,aCtcutoffpointwasdetermined,beyond whichotherCtvaluescouldnotberegardedaspositive(Kokkinos andClark,2006).

2.6. Statisticalanalysis

ThedatawasanalysedbyAnalysisofVariance(ANOVA)andthe averageofeachtreatmentwascomparedbyTukeyTest(p≤0.05).

3. Results

3.1. DetectionofPMeVinpapayaleavesbyconventionaland real-timeRT-PCR

ConventionalRT-PCRusingtheprimersPMeVconvamplifieda 300bpfragmentfrominfectedleaves(Suppl.Fig.S2).Nofragment wasamplifiedfromuninfectedsamples,confirmingthespecificity ofthePMeVconvprimersforPMeVdiagnosis(Suppl.Fig.S2).

Real time RT-PCR using PMeVreal primers presented above backgroundfluorescenceafter22cycleswhichcontinuedtogrow exponentiallyuntilreachingaplateauatcycle38.Thedissociation curvesforbothPMeVrealand28Sprimersweresharp,suggesting noprimerdimerisationornon-specificamplification(Ririeetal.,

Table1

Cycle threshold of 32samples submitted to PMeV and 28S ribosomal gene ampliﬁcation. Symptoms Sample Ct 28S PMeV Asymptomatic (FSP) 1 6.458 Undetectable 2 6.304 Undetectable 3 5.773 Undetectable 4 5.78 Undetectable Asymptomatic (CSP) 5 5.738 Undetectable 6 5.738 Undetectable 7 5.632 Undetectable 8 5.809 Undetectable 9 5.887 Undetectable 10 5.867 Undetectable 11 5.573 Undetectable 12 5.749 38.588 13 5.605 Undetectable 14 5.795 Undetectable Symptomatic 15 5.833 29.388 16 5.874 26.221 17 6.54 25.791 18 6.888 27.644 19 6.862 29.063 20 5.47 22.202 21 5.285 26.355 22 5.543 Undetectable 23 5.694 Undetectable 24 5.879 28.665 Inconclusive 25 6,909 Undetectable 26 6.672 21.735 27 6.884 Undetectable 28 6.864 Undetectable 29 6.316 Undetectable 30 6.571 Undetectable 31 6.69 Undetectable 32 6.831 Undetectable

1997).TheinfectedsamplesproducedaCtvalueof22.2while unin-fectedsamples,usedasnegativecontrol,wereindistinguishable fromthebackground(Table1).Real-timePCRampliﬁcationof28S ribosomalRNAproducedsimilarCtvalues,i.e.,5.53and5.89,from both papayastickydiseasedand uninfectedleaves, respectively (Table1).

3.2. ComparisonoffourmethodsfordiagnosisofPMeVinpapaya Thevisualdiagnosisof32papayaplantsgrowingunderﬁeld conditionswascompared tothreeotherPMeV detection meth-ods:(1)extractionoftotalnucleicacidsfromlatexsamplesand observationofthe12kbpdsRNAbandonanagarosegel(Rodrigues etal.,2005),(2)conventionalRT-PCRusingPMeVconvprimersand (3)real-time RT-PCRusingPMeVrealprimers.Aftervisual diag-nosis,theplantsweredividedinsymptomatic,asymptomaticand inconclusive.Theasymptomaticplantswerecollectedclose(CPS) orfar(FPS)fromsymptomaticplants.Thepresenceofthe12kbp dsRNAbandwasobservedinsixsamples,numbers15,17,18,19, 24 and26 (Fig.1A).Five ofthesesampleshad beendeﬁnedas symptomaticandoneinconclusive.Fivesymptomaticplantsdid notpresentbands(Fig.1A).

Conventional RT-PCR method using PMeVconv primers detectedPMeVinnineleafsamples,eightsymptomaticandone inconclusive(Fig.1B).Theseincludedalltheplantsdiagnosedby 12kbpdsRNAdetection,butalsosymptomaticplants,numbers16, 20 and21, demonstrating greatersensitivity.Twosymptomatic plantswereclassiﬁedasvirusfreeby12kbpdsRNAdetection,but noasymptomaticplantswerediagnosedasinfected.

TheCtvalues obtainedbyreal-timePCRusingthePMeVreal primersarepresentedinTable1.ThesamplespositiveforPMeV

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Fig.1.ComparisonofthreemethodsfordiagnosisofPMeVinpapaya.Papayaplants(n=32)werevisuallygroupedassymptomatic,asymptomaticandinconclusivefor papayastickydisease.Asymptomaticplantsampleswereharvestedfar(FPS)orclose(CPS)todiseasedplants.ThepresenceofPMeVwasfurtherassessedbytwomolecular detectionmethods,theextractionofPMeV12kbpdsRNAfrompapayalatex(A)andtheampliﬁcationofPMeVRDRPgenebyconventionalRT-PCRusingPMeVconvprimers (B).Theampliﬁedfragmentswereseparatedby1%(A)or1.5%(B)TBEagarosegels,stainedwithethidiumbromide.M,molecularweightmarker.

byreal-timeRT-PCRwerethesamethattestedpositiveby conven-tionalRT-PCR(Table1andFig.1B).Thus,ofthetenplantsdiagnosed asinfectedbyvisualsymptomsinthefield,eightwereconfirmed byconventionalandreal-timeRT-PCR.Alltheplantsdiagnosedas asymptomaticwereconfirmednegativeforPMeV.

3.3. AssessmentofPMeVtransmissionbypapayaseeds

Twogroupsofseedlings15–20daysaftergerminationwere ana-lysed:seedlings(n=187)fromseedscollectedfromhealthypapaya fruits,andseedlings(n=172)fromseedscollectedfromdiseased fruits.ThepresenceofviruswasassessedinpooledRNAsamples fromeachplantgroupbyconventionalRT-PCRusingPMeVconv primers.BothseedlinggroupstestednegativeforPMeVpresence (Fig.2),suggestingthevirusisnottransmittedbyseeds.The ampli-ﬁcationofanactingene,usedasapositivecontrolfortheRT-PCR reaction,wasobservedforallsamples(Fig.2).

3.4. Co-inoculationofpapayawithPMeVandpuriﬁedPMeV dsRNA

Inorder toassesstheeffectof purifiedPMeV dsRNAonthe progressofPMeVinfectioninpapaya,theplantswereco-inoculated withPMeVinfectedlatexandPMeVdsRNA.Plantsinoculatedwith phosphatebufferpH7.0orPMeVinfectedlatexwereusedas nega-tiveandpositivecontrols,respectively.Thevirusloadintheplants wasestimatedusingthreedifferentmoleculardiagnosismethods, i.e.,extractionofPMeV12kbpdsRNAfrompapayalatex, amplifica-tionofPMeVRDRPgenebyconventionalRT-PCRusingPMeVconv primersandreal-timeRT-PCRusingPMeVrealprimers.Thelatexof thoseplantsco-inoculatedwithpurifieddsRNAandinfectedlatex presentedthePMeV12kbpdsRNAband43dpi(Fig.3A).The posi-tivecontrolplantsclearlypresenteddsRNA29dpi.Thesedatawere

Fig.2.Evaluationof PMeVtransmissionthrough papayaseeds.Papaya seeds (n=172)collectedfromdiseasedfruitweregerminatedinPetridishes.Theseedlings at15–20dayspostgerminationwerepooledandsubmittedtoRNAextraction.Seeds (n=187)fromhealthypapayafruitwereusedasnegativecontrol.DuplicateRNA samplesfromthecontrol(1–2and5–6)andfromthegerminatedseedsfrom dis-easedfruit(3–4and7–8)wereanalysedbyconventionalRT-PCRusingPMeVconv primers.TheampliﬁcationofactingenewasusedasaRT-PCRreactioncontrol. Thenucleicacidswereseparatedon1.5%TBEagarosegels,stainedwithethidium bromide.M,molecularweightmarker.

conﬁrmedthroughtheanalysisofleafsamplesbyconventional RT-PCR using PMeVconvprimers (Fig. 3B) and suggest the co-inoculationofpapayaplantswithpuriﬁedPMeV-dsRNAdelayed theinfectionofPMeV.TheloadofPMeVinco-inoculatedsamples appearedreducedat57dpi(Fig.3AandB),suggestingthatPMeV dsRNAcouldalsoelicitgeneraldefencesinpapayaplants,which wouldresultintheinhibitionofvirusreplication.

Theseedlingsinoculatedonlywithbufferalsoshowedafaint bandat22and57dpi,possiblyasaresultofaminorinfectionduring theexperiment.

Thelatexsamplescollectedfromseedlingswerealsoanalysed byconventional RT-PCR usingtheCO5-3 primer(Araujo etal., 2007),butallsamplestestednegative(Suppl.Fig.S3A).Incontrast, whennucleicacidswereextractedfromlatexofstickydiseased matureplants,theexpectednucleicacidbandof200–300bpwas clearlyseen(Suppl.Fig.S3B),verifyingthattheprimerswere func-tional.TheseresultssuggestthatRT-PCRusingCO5-3 primeris moresuitablefortheanalysisofpapayalatexfrommatureplants withhighvirustiters.

InordertoestimatethePMeVloadintheco-inoculatedpapaya plants, the samples were analysed by real-time RT-PCR using PMeVrealprimers.Forrelative quantification,thedataobtained foreach sample wasnormalisedagainst theribosomal28S ref-erencegene. Bothgroups ofplants, co-inoculatedwithpurified PMeVdsRNAanddiseasedlatexandpositivecontrols,presentedan increasingvirusloadovertimewhichwasclearlylesspronounced intheco-inoculatedplants(Fig.4).Thesedatafurtherconfirmthe delayedPMeVinfectioninpapayainoculatedwithpurifiedPMeV dsRNA.

Fig.3. AssessmentofPMeVloadinpapayaplantsco-inoculatedwithPMeVand puriﬁedPMeVdsRNA.Theplantswereco-inoculatedwithPMeVinfectedlatex andPMeVdsRNA(PMeV+dsRNA)orinoculatedonlywithphosphatebufferpH7.0 (Buffer)orPMeVinfectedlatex(PMeV).Thelatexsamplescollectedfromeachplant atregulardays-postinoculation(dpi)wereanalysedbyextractionofPMeV12kbp dsRNA(A)ortheampliﬁcationofPMeVRDRPgenefrompapayaleafbyconventional RT-PCRusingPMeVconvprimers(B).Theresultantmaterialwasseparatedby1%(A) or1.5%(B)TBEagarosegels,stainedwithethidiumbromide.M:molecularweight marker.

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Fig.4. AssessmentofPMeVloadinpapayaplantsco-inoculatedwithPMeVand puriﬁedPMeVdsRNAbyreal-timeRT-PCR.RelativeabundanceofPMeVinpapaya samplescollectedfromplantsinoculatedwithPMeVinfectedlatex(openbars), co-inoculatedwithPMeVinfectedlatexandPMeVdsRNA(diagonallines)and inoc-ulatedwithphosphatebuffer(blackbars).Sampleswerecollectedatdifferentdays postinoculation(dpi)andampliﬁedusingPMeVrealprimer.

Theco-inoculatedplantstestedpositiveforPMeVat50and29 dpiwhenanalysedbyregularRT-PCR(Fig.3B)orreal-timeRT-PCR (Fig.4),respectively.Theseresultsindicatethegreatersensitivityof thereal-timeRT-PCRmethod,asalreadyreportedforotherviruses (KokkinosandClark,2006).

4. Discussion

ThediagnosisofPMeVisofprimaryimportanceinthecontrol ofpapayastickydiseasegiventhatattimeofwritingthereareno commerciallyavailableresistantcultivars.Diagnosisisprimarilyby assessmentofsymptoms.

Visualdiagnosisisrelativelyquickandcheap,butithasa num-berof weaknesses as a technique. Unfortunately, for unknown reasons,symptomsonlybecomeapparentafterflowering(Ventura etal.,2004)andintheirearlieststagesareopento misinterpre-tation. Additionally, in thefield, the first symptoms of papaya stickydiseasearepresentedonly45daysafterinoculationwith PMeV infected latex (Ventura et al., 2004).This window of 45 daysbetweenvirusinfectionandtheonsetofsymptomsplusthe non-appearanceofsymptomsthroughouttheperiodpriorto flow-eringcanleadtothepresence ofasymptomaticplants infected withPMeV.Theseasymptomaticplantscanprovideahigh concen-trationofthevirusforsubsequenttransmission,sincethePMeV dsRNAisalreadydetectable15daysafterinoculationofthevirus inpapayaplants(Rodriguesetal.,2005).Highairtemperaturecan alsoaffecttheappearanceofsymptomsofpapayastickydisease (Venturaetal.,2004).Therefore,plantscanharbortheviruswithin anorchard,actingasreservoirsforinfection.

Twomethods have been publishedfor assessment of PMeV infection,i.e.,detectionofviral12kbpdsRNAusingagarosegels andethidiumbromide(Rodriguesetal.,2005)andconventional RT-PCRonnucleicacidsfromcrudelatex(Araujoetal.,2007).Thefirst methodhasadvantagesoverothermethodofbeingrelativelyrapid andlowercost.However,variousstudieshavereportedarestricted detectionofnucleicacidsinagarosegelsstainedwithethidium bromide.ThedetectionlimitforaviraldsRNAbandisestimated tobe100ng(Mcfaddenetal.,1983).Thus,sincethedetectionof purifiedPMeVdsRNAdoesnotincludeanyamplificationstep,the quantityofviraldsRNAextractedmustexceedthisdetectionlimit. Incontrast,10fgcanbesufficientfordetectionofviralgenomeby RT-PCR(RomaineandSchlagnhaufer,1995).Afurther considera-tionisthatdiagnosisofPMeVbydirectapplicationofpurifiedviral dsRNAtoanagarosegelcanresultinfalsepositivesforthevirus, duetotheproximityofmolecularweightsofPMeVdsRNA(12kbp) andpapayaDNA(∼14kbp).

RT-PCRhasbeenusedsuccessfullyinthedetectionofa num-berofplantviruses(Mekuriaetal.,2003;Omunyinetal.,1996) and hasbeen shown tobe 103_–104 _times _more _sensitive_than

immunoassays, analternative technique (Zhu et al., 2010).The methodofPMeVdetectionbyRT-PCRproposedpreviously(Araujo etal.,2007)usescrudepapayalatexdilutedin0.1Mammonium cit-rate(1:1,v/v)withoutpriorextractionofnucleicacidsastheinitial material.ThepresenceofPMeVinthepapayalaticifers/latexiswell established(Kitajimaetal.,1993;Venturaetal.,2004;Zambolim etal.,2003)andthereforelatexsamplesaretheprimarychoicefor virusdetection.Animportantpointtobeconsideredisthehigh con-centrationofproteinspresentinpapayalatex(Moutimetal.,1999) whichcanresultinlowyieldinRT-PCR.Astronglinkbetweenviral particlesandlatexpolymerhasbeenobserved(Rodriguesetal., 2009c)thatcandecreasetheamountofdsRNAPMeVavailablefor reversetranscriptasereaction.Additionally,therearesomestages ofdevelopmentofpapayaforwhichevenasmallamount(e.g.,5␮L) isunavailable,suchasyoungseedlings.

Seedlings present challenges in the collection of latex. The amountexudedisverysmall,andisrelatedtoturgescence pres-surewithinthelactifer(Moutimetal.,1999).Theuseofasmall amountoflatexcanbedisadvantageousforviraldiagnosisdueto insufﬁcientnucleicacidforanalysis,especiallyearlyinaninfection (JarosovaandKundu,2010),thusgeneratingfalsenegatives.

Inthisstudy,latexsamplescollectedfrommaturepapaya,or papaya seedlings previously inoculated with PMeV, were ana-lysedusingthemethodpreviouslydescribed(Araujoetal.,2007). However,onlylatexfrommatureplantstestedpositive.Thus,the analysisoflatex bythismethoddoesnotappeartobesuitable fordiagnosisofPMeVinseedlingsbecauseofthelowvirustiter. TheRT-PCRmethoddevelopedusingtheprimersPMeVconvdid detectPMeVfromseedlingleavesinoculatedwiththevirus.Using leafmaterialappearstobepreferableforvirusdetectioninpapaya seedlingsbecauseofthegreaterpurityofmaterialextracted.

Theconventional RT-PCRdetection methodproposedin this paperusesabout100ngofpurifiedRNAasatemplatefor ampli-fication, a quantity achievable from seedlings. This amount is approximatelythesameasusedforRT-PCRdetectionofPrunus necroticringspotvirusinalmond(Mekuriaetal.,2003)andfor sev-eralstrainsofSoybeanmosaicvirus(Omunyinetal.,1996).Although conventionalRT-PCRwassuccessfulindetectinglowtitersofPMeV insmallquantitiesofmaterial,amethodforreal-timeRT-PCRwas alsodeveloped.Thishastheadvantageofgreatersensitivity,the simultaneoustestingoflargenumbersofsamples,automated anal-ysisoftheresultsandreliablequantification(Beuveetal.,2007;Lim etal.,2010).

Therearetwogeneralapproachesforthedetectionofamplified fragmentsbyreal-timeRT-PCR,theuseofspecificandnon-specific fluorescentreporters.Bothhavesimilarlevelsofsensitivity(Bustin and Nolan, 2004).However, theuse of specificprobes suchas TaqMan canresultinfalse negatives,especially in RNAviruses. Alternatively,non-specificprobessuchasDNAintercalatingdye SYBR Green,are morereliable,simple andlow cost(Jamesand Varga,2005;Papinetal.,2004).Althoughverysensitive,real-time RT-PCRrequiresacutoffpointfordetectiontopreventfalse pos-itives.ThemaximumvalueofCtthatisconsideredpositivefora particularvirusvaries.WhileCtvaluesupto45havebeenaccepted asvalidforrhinovirusdetection(Wadowskyetal.,2010),in gen-eral35isconsideredanegativeresult,forexamplefordetection ofSweetleafcurlvirusinsweetpotato(KokkinosandClark,2006). Thus,itisquestionablewhethersample12canbeconsidered pos-itivewithaCtof38.58withouttheestablishmentofastandard curveofvirusconcentrationversuscyclethreshold.Therefore,we preferredtoconsidertheresultofthissampleasinconclusive.

Althoughreal-timeRT-PCRismoresensitivethanconventional RT-PCR,thetwomethodsshowedsimilarresultsinvirusdetection inmatureplants,perhapsbecausethediseased plantscollected containedarelativelyhighvirustiterwithinthedetectionlimits ofbothtechniques.BothPCRtechniquesweremoresensitivethan

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directobservationofthevirus12kbpdsRNAgenomeonagel.Both alsohavetheadvantagethattheycanbeappliedtoalllifestagesof papaya,andnotjusttoadultplantsafterﬂowering.Thedetectionof virusbyreal-timeRT-PCRalsoallowsforthequantiﬁcationofPMeV whichcouldcontributetofutureexperimentsaddressingthevirus biology.

Thequestionoffalsepositiveandnegativesisofcrucial impor-tance in PMeV control. Of the 10 plants diagnosed by visual symptomsinthefieldonly8wereconfirmedbythetwo meth-odsofPCR.Thesefalsepositivescanoccurastheinitialsymptom inthefield isthenecrosis oftheleavestipswhich canalsobe causedbythefungusStagonosporopsiscariacae(Syn:Phoma caricae-papayae)ordamageresultingfrominsectinfestation(Venturaetal., 2004).Intermsoffalsenegatives,alloftheplantsdiagnosedas asymptomaticwereconfirmedbyPCR.However,oneoftheplants diagnosedvisuallyasinconclusivewasdiagnosedaspositiveby PCRmethods.Inpractice,plantsarenotnormallyremovedfrom cultivationunlesspositivelyidentifiedasinfected.Plantsleftinthe fieldcanclearlyserveasavirusinoculumforotherplants.

Anadditionalproblemisunderstandingtransmissionofpapaya stickydisease.Withmanypapayaplantsgrownfromseeds,such plantsarealsoapotentialsourceofinfection.Ithasbeenunclear whetherPMeV can betransmitted from parenttoseedling via seeds. Thiswas therefore investigated,as well asdeveloping a methodfordiagnosisofvirusinfectioninseedlings.NoPMeVwas detectedbyconventionalRT-PCRusingthePMeVconvprimerpair onpoolsofseedlingsderivedfromseedsofhealthy(n=187)and infected(p=172)plants,respectively.However,although sugges-tive,theseresultsdonotprovethelackoftransmissionofPMeV byseeds. Thefrequency of transmission of PMeV maybe very low,requiringa largernumber ofseedlingstobeanalysed.The frequencyofvirustransmissionbyseedscanvarygreatly depend-ingonthevirus-hostinteraction,andmaybeverysmallinsome cases.AlthoughCocoaswollenshootviruscouldbedetectedin53 of98cocoaseedlingsanalysed(Quainooetal.,2008)andCucumber mosaicviruswasdetectedin27of180seedlingsinvestigated(Yang etal.,1997),HighPlainsviruswasdetectedinmaizeinonlythree of38,473seedlingsanalysed(Forsteretal.,2001).Similarresults wereobtainedinthetransmissionofMaizedwarfmosaicvirus,in whichonlyoneof22,189seedlingswasinfected(Mikeletal.,1984). Soalthoughvirustransmissionbyseedscannotberuledoutbythis work,itissuggestedthatifisoccurringitiswithlowfrequency.

Ashasbeenmentioned,there arenocommerciallyavailable PMeVresistantpapayacultivars.Ithasbeensuggestedinthe lit-eraturethatplantscanbeinoculatedwithviraldsRNAasawayof preventingordelayinginfectionandthishasbeendemonstrated invivo(Tenlladoetal.,2003).Thus,ourresultsareinaccordance withothersintheliteraturewhichshowedadelayofviral infec-tion by the application of dsRNA sequences (Gan et al., 2010; Tenlladoetal.,2003),possiblyviaposttranscriptionalgene silenc-ing(Tenlladoand Diaz-Ruiz,2001).Perhapssurprisingly,dsRNA appliedexogenouslycanpersistontheleafdespitewateringand rain(TenlladoandDiaz-Ruiz,2001).Thisprincipleappearstobe worth further consideration as a method for PMeV control in papaya.

Conﬂictofintereststatement

Theauthorsdeclarenoconﬂictofinterest.

Acknowledgments

TheauthorsthankProfessorAdrianaSilvaHemerly (Universi-dadeFederal doRio deJaneiro)for herassistancewiththereal timeRT-PCRanalysis.ThisworkwassupportedbyFinanciadorade

EstudoseProjetos(FINEP),ConselhoNacionaldeDesenvolvimento CientíﬁcoeTecnológico(CNPq),BancodoNordestedoBrasil(BNB), andtheFundac¸ãodeAmparoàPesquisadoEstadodoEspíritoSanto (FAPES).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.jviromet.2011.12.003.

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